GldA

Glycerol dehydrogenase GldA - Overview

The Gene GldA encodes glycerol dehydrogenase from Escherichicha coli. The overexpression of glycerol dehydrogenase in E. coli can be used as a way for endogenous mediator production. Many derivates of glycerol dehydrogenase are small, water-soluble redoxmolecules, which have mediator properties. In enzymology, a glycerol dehydrogenase (EC 1.1.1.6) is an enzyme belonging to the family of oxireductases that catalyzes the chemical reaction: Glycerol + NAD+ <--> Glycerone + NADH + H+ with NADH as the main endogenous mediator.

Theory

• Theory for GldA glycerol dehydrogenase.

Genetic Approach

• The GldA gene from Escherichia coli was cloned and heterologously expressed in E. coli KRX under the control of different promoters (Table 1).

Table 1: Overview of GldA devices. Combination of GldA coding BioBrick (<bbpart>BBa_K1172201</bbpart>) with different promotors and RBS.

Figure 1: pSB1C3 – <bbpart>BBa_K1172201</bbpart> GldA BioBrick (1104 bp) was examined by restriction analysis and sequencing.

Results

• Upon the expression of the glycerol dehydrogenase, the endogenous mediator production of Escherichia coli was measured. SDS-PAGE combined with MALDI-TOF MS/MS and different NADH-assays characterize GldA BioBrick <bbpart>BBa_K1172201</bbpart>.

SDS-PAGE and MALDI-TOF

• SDS-PAGE shows protein Glycerol dehydrogenase at expected size of 40 kDa. In contrast to Escherichia coli KRX Wildtyp, weak Anderson promoter (<bbpart>BBa_K1172205</bbpart>) shows only a slightly stronger band, whereas T7 (<bbpart>BBa_K1172203</bbpart>) and Lac (<bbpart>BBa_K1172204</bbpart>) promotor show a strong band, which is equated with a strong expression and overproduction of GldA.

Figure 2: SDS-PAGE with Prestained Protein Ladder from Thermo Scientific as marker. Comparison of protein expression between Escherichia coli KRX wild type and Escherichia coli KRX with <bbpart>BBa_K1172205</bbpart>, <bbpart>BBa_K1172204</bbpart> and <bbpart>BBa_K1172203</bbpart> after total cell disruption. The right-hand gel was loaded with a higher protein concentration. SDS-PAGE shows protein Glycerol dehydrogenase at expected size of 40 kDa. Enhanced overproduction with increasing promotor strength.

• Furthermore we were able to identify the overexpressed glycerol dehydrogenase (Figure. 2) with MALDI-TOF MS/MS.
  • Tryptic digest of the gel lane for analysis with MALDI-TOF could examine the glycerol dehydrogenase with a Mascot Score of 266 against Escherichia coli database.

NADH-Assays

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References

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